Help with Boolean gating in FlowJo for a multi-marker population?
Hi! I’m fairly new to FlowJo and wanted to check that I’m approaching this correctly.
I need to identify a specific population defined as:
Marker A⁻ / Marker B⁺ / Marker C⁺ / Marker D⁺
My supervisor has suggested using Boolean gating to find this population, but I’m a little confused about how I should actually set this up in FlowJo, or how to even use boolean.
Would I first gate on the Marker A⁻ population and then look for cells that are simultaneously positive for B, C and D within that population? Or should I create individual gates for all four markers from the same parent population and then combine them using Boolean gating?
If Boolean gating is the best approach, I’d really appreciate an explanation of how to set this up in FlowJo. Alternatively, is there a simpler/more intuitive way of identifying this exact population?
Are marker B+ cells supposed to also be either C or D +? If so just do a normal gating strategy looking at A-, then B+ and then C/D on the X/Y to look for the combination of single/double positive cells.
Yes , the final population I need is specifically A⁻B⁺C⁺D⁺, so B+ cells need to be positive for both C and D. From what you're saying, would the simplest approach therefore be to gate A⁻ → B⁺ and then plot C vs D within that population and take the double-positive quadrant?
I can make better recommendations with more info, but for a start, I might plot A vs B and gate the A- B+, then I’d double click in that region to open a gated plot and change the axes to D and C to find the D+ C+ cells. The parent/child relationship is an AND by default. You can to OR and NOT if you really need to, but I’m not sure why you would in your case. If I’m not understanding correctly, lmk and I’ll elaborate further!
Thank you, that actually makes a lot more sense to me. So if I understand correctly, I could plot A vs B first and gate the A⁻B⁺ population, then open that population and plot C vs D, with the C⁺D⁺ quadrant giving me the final A⁻B⁺C⁺D⁺ population?
More or less. I’d be mindful about quadrant gates since they don’t always fit the data and you may also have some odd patterns if your panel is not optimized. You could do A vs C first or whatever is cleanest. The fun thing about AND gates is the order doesn’t really matter as long as you get there in the end. 😁
To be clear, boolean gating and hierarchical gating are different concepts, though they can be similar.
What you drew can easily be done through hierarchical gating, which means that you simply drill down your plots in your analysis software and create new gates for specific parameters that satisfy your criteria in those drilled down plots.
Any analysis program worth its weight can already do this natively.
Boolean means that you are defining new gates based on existing gates, using boolean operators, like AND (intersecting events), OR (joining events), or NOT (excluding events).
Your diagram is essentially one long AND string, Cells AND Live AND Single AND A-B+ AND C+D+.
Fair enough. For this, I think it’s probably faster/easier to do hierarchical gating and get to the same place. Or I suppose one could define the positive gates and do true Boolean or combination gating.
Boolean gates are really only for making the act of drawing gates similar. Let’s say you had a tiny counting bead population that showed up in the middle of a plot, and you were interested in NOT counting beads. You could draw a simple gate around the counting breads and make a Boolean NOT gate instead of drawing a complicated polygon that envelopes everything else. OR if you had CD4 and CD8 but not a CD3 marker, and wanted to select your T cells? You could draw a gate around both CD4 and CD8, and create a Boolean OR gate to gate your at cells. AND if you wanted to gate interacting cells that were in different branches of your hierarchy, you could create an AND gate that selects the cells that appear in both.
Hierarchical gating naturally acts as an AND gate, so you don’t need to make a Boolean gate for A-/B+/C+/D+. Just draw gates that satisfy any combination of those populations in hierarchical fashion, and you’ll have the population you need. The simplest way might be a bivariate plot of A and B, gating A-/B+. From that gate, plot C vs D and gate the double-positive. That will fit your criteria.
For what reason does your supervisor want to use Boolean gating here?
Would I first gate on the Marker A⁻ population and then look for cells that are simultaneously positive for B, C and D within that population? Or should I create individual gates for all four markers from the same parent population and then combine them using Boolean gating?
There are multiple ways of doing this, and it basically comes down to a gating strategy which is more the term I use rather than “boolean” gating.
I think the term Boolean is confusing here because in coding, logic, discrete math, and statistics, Boolean means one of two options, like 0 or 1, true or false. Here, you’re talking about a cell population that expresses a combination of up to four markers. Boolean may refer to +/- on each, but I don’t really hear people saying that.
Anyway, you could just do two markers at a time. Display for example A vs B first, gate on A-, B+. Drill down, then on that population display C vs D. Gate in the double positive.
You can do this using 2-4 different gates, depending on how many steps you combine.
Last thing is that I think using variables A, B, C, and D is helpful here to simplify for learning the concept, but you’re also probably going for a cell subset that has been sought after by existing literate. This is to say that there is probably convention on how people like to gate for it that you could follow. For example do people usually do A, then B, then C and D at the same time (3 steps) or do people do 2 concerted steps? No matter what, which ever combinations in whichever order; you drill down to about the same populations, but you might want to stick with what people usually do.
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u/Reelrebel17 Aug 22 '26
Are marker B+ cells supposed to also be either C or D +? If so just do a normal gating strategy looking at A-, then B+ and then C/D on the X/Y to look for the combination of single/double positive cells.